Imaging of drug accumulation as a guide to antitumor therapy

ABSTRACT

The present invention describes the use of radio-labeled antitumor drugs in the treatment of solid tumors by the method of administering a radio-labelled anticancer drug to a patient and imaging at least a part of the patient using Positron Emission Tomography imaging. The method is used to monitor delivery of antitumor drugs to tumors and may be used to predict the effectiveness of therapy with a particular antitumor drug or combination of antitumor drugs, to assess the effectiveness of modulators of cellular accumulation, to individualize therapy and to evaluate the effectiveness of antitumor drugs with respect to particular cancers. Particularly preferred drugs are labeled taxanes, e.g.,  11 C-paclitaxel and  11 C-docetaxel, labeled anthracyclines, e.g.,  11 C-doxorubicin and  11 C-epirubicin, and other radio-labeled drug, e.g.  11 C-topotecan and  11 C-mitoxantrone. The invention further describes antitumor drugs labeled with the radioactive label  11 C and methods of preparing radio-labeled drugs.

This application is a 371 national stage of international applicationNo. PCT/US00/25833, filed Sep. 21, 2000, which claims benefit of U.S.provisional application No. 60/155,061 filed Sep. 21, 1999.

FIELD OF THE INVENTION

The present invention relates to the use of radio-labeled antitumordrugs in the diagnosis and treatment of cancers characterized by solidtumors. The invention also relates to radio-labeled antitumor drugs andtheir preparation.

BACKGROUND

Cancer treatment has made great progress in recent years. Many newtherapies are becoming available, and many more patients are treated.Cancer is characterized by unrestricted cell growth; many cancertherapies work by inhibiting cell division. Since normal cells do notdivide after maturation, inhibition of cell division primarily affectsthe cancer cells and this has been a focus of drug development. However,other cells are also affected by antitumor drugs to different degreesand cancer therapies are often extremely toxic to patients. There isalso great variability in the efficacy of treatments. Some drugs aremore effective than others for certain patients, for certain cancers, orat certain stages of treatment. Often, combinations of drugs withvarying dosages are necessary for efficacious treatment, requiringconsiderable experimentation to optimize drugs and doses.

Among the agents affecting the therapeutic benefits of cancer drugs arethe multi-drug resistance transporter (MDR), also called p-glycoprotein(PGP) or ABCB 1. It has recently become clear that MDR is one of afamily of such transporters. Klein I, Sarkadi B, Varadi A. An inventoryof the human ABC proteins. Biochim. Biophys. Acta 1999; 1461:237–62. Theexpression of these proteins can be highly variable. MDR is notexpressed in all cancer cells, and may be present at variable levels.The expression of MDR affects drug efficacy by altering drugaccumulation at the tumor. Because of the variable efficacy of antitumordrugs caused by this and other factors, a critical part of therapeuticmonitoring involves determining the drug location in the body, itshalf-life, and the range of mechanisms that limit its effectiveness.

Accumulation of a drug reflects the net balance over time of influx(delivery to the tumor) and efflux (removal from the tumor). Influx andefflux are equally important, but recent research has focused upon aseries of transport proteins that function as efflux pumps for taxanes,anthracyclines, and other drugs. There are many reasons why a tumor maynot be sensitive to a particular drug. However, the first parameter toevaluate is accumulation of drug by the tumor. If the drug doesn'taccumulate in the tumor, there won't be an effect. Adequate accumulationis always necessary for drug activity. Thus, a method to determineaccumulation of a drug in the tumor could be the first step intherapeutic decision-making for the drug.

Traditional approaches to the determination of drug uptake and retention(drug accumulation in the tumor) have been invasive and most frequentlyrequire obtaining a biopsy from the patient. In addition to thediscomfort and risks associated with biopsy procedures, only a smallsample of tissue is typically obtained, which may not be representativeof the entire region.

Taxanes

One class of drugs which has proved particularly useful in the treatmentof cancer, including solid tumors such as breast cancer, has been thetaxanes. Taxanes are diterpenoid compounds with a complex taxane ring asthe nucleus. The taxane paclitaxel (I) (Taxol®) was initially isolatedfrom yew bark, although the compound may now be prepared synthetically.A modification of the side chains of paclitaxel has yielded anotherclinically effective compound, docetaxel (II). Other taxanes have alsobeen developed, and are at various stages of preclinical and earlyclinical testing. These analogs include differences in functional groupsattached to the main baccatin nucleus, as well as different side chainsattached at the C-13 position.

-   -   (I) Paclitaxel: R₁=R₂=acetate, R₃=Ph    -   (II) Docetaxel: R₁=OH, R₂=acetate, R₃=OC(CH₃)₃

Taxanes, like the vinca alkaloids and colchicine, work by interferingwith microtubules, thereby inhibiting mitosis. Taxanes antagonizedisassembly of microtubules, by promoting tubulin polymerization,inducing microtubule bundles to form. This leads to arrest of mitosis,and ultimately to cell death. The rate of cell death is proportional toconcentration of drug and length of time of administration. Taxanes arehighly insoluble and are commonly administered in a solution ofsurfactants and other vehicles such as ethanol. (Hardman, J. G. andLimbird, L. E., (eds) Goodman and Gilman's The Pharmacological Basis ofTherapeutics, Chapter 51, McGraw-Hill, New York, 1996)

The taxane class of anticancer drugs has demonstrated remarkableactivity. Docetaxel and paclitaxel, the first two approved drugs in thisclass, have already altered the standard treatments for breast, lung,and ovarian tumors. Crown J, O'Leary M. The taxanes: an update. Lancet2000; 355(9210):1176–8. Burris H A 3rd. Docetaxel (Taxotere) in thetreatment of cancer. Semin. Oncol. 2000; 27 (2 Suppl 3):1–2. The fullscope of antitumor activity for docetaxel and paclitaxel is still underactive investigation, with new uses emerging for other tumor types. Forexample, at present there is considerable interest in the use ofdocetaxel for treatment of prostate cancer. Oh WK, et al. Docetaxel(Taxotere)-based chemotherapy for hormone-refractory and locallyadvanced prostate cancer. Semin. Oncol. 1999; 26 (5 Suppl 17):49–54;Petrylak D P. Docetaxel (Taxotere) in hormone-refractory prostatecancer. Semin. Oncol. 2000; 27 (2 Suppl 3):24–9. Other molecules in thetaxane class are at much earlier stages of clinical testing.

Anthracyclines

Anthracyclines represent another important class of antitumor drugs.Representative anthracycline drugs include doxorubicin (III) andepirubicin (IV). These anthracyclines are leading agents for thetreatment of many tumors, notably breast cancer, lung cancer, andsarcomas. Doroshow J H. Anthracylines and anthracenediones. In: ChabnerB A and Longo D L, Cancer Chemotherapy and Biotherapy, 2^(nd) Edition,p. 409, Lippincott-Raven, Philadelphia, 1996. Several mechanisms ofaction have been proposed, based upon the intercalation of theanthracycline molecules with DNA, and subsequent disruptions of cellularfunctioning.

-   -   (III) Doxorubicin: A=H; B═OH    -   (IV) Epirubicin: A=OH; B═H

Other Antitumor Drugs

Because the discovery of anticancer drugs has been well-funded over thelast half-century, drugs from a variety of chemical classes are now inroutine clinical use. Mitoxantone (V) is an anthracenedione, a chemicalclass closesly related to anthracyclines. Mitoxantrone was originallydeveloped in an attempt to replace anthracyclines because theanthracenediones have lower cardiac toxicity. However, the antitumoractivity of mitoxantrone was generally disappointing and it has arelatively narrow niche in clinical use. Recently, however, mitoxantronehas been demonstrated to substantially reduce the severe pain associatedwith metastatic prostate cancer, and has changed the management of thislarge group of cancer patients. Tannock I F, Osoba D, Stockler M R,Ernst D S, Neville A J, Moore M J, Armitage G R, Wilson J J, Venner P M,Coppin C M, Murphy K C. Chemotherapy with mitoxantrone plus prednisoneor prednisone alone for symptomatic hormone-resistant prostate cancer: aCanadian randomized trial with palliative end points. J. Clin. Oncol.1996; 14(6):1756–64.

-   -   (V) Mitoxantrone

Camptothecin is a natural product found in the bark and wood of aChinese tree. Although it was found to be too difficult for clinical useitself, a number of derivatives have demonstrated clinical activity.Takimoto C H, Arbuck S G. The Camptothecins. In: Chabner B A and Longo DL, Cancer Chemotherapy and Biotherapy, 2^(nd) Edition, p. 463,Lippincott-Raven, Philadelphia, 1996. This class of drugs appears towork by inhibiting the action of topoisomerase I, a key enzyme for theintegrity of DNA structure. Irinotecan (CPT-11) was initially approvedfor treatment of colorectal cancer, and topotecan (VI) initiallyapproved for

-   -   (VI) Topotecan        ovarian cancer. Both of these two drugs and other camptothecin        analogs are acquiring new uses as testing continues. Topotecan        has shown activity in lung cancer, and is now also approved for        treating these patients. In addition, topotecan is particularly        important from the perspective of PET imaging because it is        direct-acting (irinotecan is a prodrug that must be activated),        and also has limited catabolism, so that the parent molecule is        the principal circulating species.

Determination of Sensitivity or Resistance

Although drugs having excellent antitumor activity e.g. taxanes,anthracyclines and others, have been identified, not all tumors respondto a given therapy. Furthermore, all patients are exposed to the risk ofsevere, life-threatening toxicity with these drugs whether or not theirtumors respond. An important tool for individualizing therapy would be amethod to rapidly determine whether a specific tumor will be likely torespond to a particular drug without exposing the patient to toxiclevels of the drug.

In addition to avoiding needless toxicity, a rapid determination is alsoimportant because of the tendency of tumors to become more difficult totreat with time. Thus, if one treatment can be predicted to beunsuitable, alternative treatments can be explored without waitingmonths to determine that the first treatment did not work.

In some cases, the cause of treatment failure can not be determined. Inother cases, a specific biochemical or molecular mechanism can beascertained. For example, the tumor cells may be intrinsically sensitiveto the drug, but inadequate amounts of drug are accumulated in thetumor. There are multiple reasons for drug accumulation failures, but,regardless of the underlying cause, the empirical demonstration thatadequate (or inadequate) drug was accumulated has enormous medical valuein terms of treatment selection for individual patients. Determiningdrug accumulation levels in a specific tumor pre-treatment would thusprovide a great value in exploring a wide range of treatment options.

In addition to the benefits for individual patients, determination ofdrug delivery has benefits for the general patient population and forthe process of drug development. General patient populations include amixture of tumors which are chemosensitive and chemoresistant. Thedemonstration of a substantial effect is made difficult when theresponding tumors are diluted in a pool of nonresponding tumors. Thus,any technique which can find tumors likely to respond before treatmenthas begun will produce an enriched study population, and greatlydecrease the numbers of patients required to test the overall potentialbenefit of the drug.

Position Emission Tomography (PET) imaging or scanning uses positronemitter labeled tracers. Positrons are positively charged electronswhich result from the decay of a proton rich and neutron deficientisotope. These emitters are generally short lived. Most positronemitters are produced in medical cyclotrons or accelerators. The halflife of ¹¹C is 20 min and of ¹⁸F is 110 minutes. PET cameras have aspatial resolution of several millimeters and can be used to image theentire body.

The concept of trying to find radiolabeled probes that would be idealfor the investigation of one or more transport pumps should not beconfused with the concept of measuring accumulation of the specific drugto guide therapy. The literature is filled with research that attemptsto define drug transport systems. Many groups are attempting to find the“ideal” probe for each transporter. The emerging problem is an explosionin the numbers of transporters that are being discovered. Thus, attemptsto guide therapy with a drug based upon ideal probes is fraught withdifficulty and confounded by the multiplicity of transport mechanisms,which will vary from tumor-to-tumor.

Clinical attempts to measure drug accumulation with imaging have beenreported, including the use of ¹¹C-verapamil, ¹¹C-daunorubicin, or^(99m)Tc-sesta-MIBI. All have been limited because they only target MDRand have additional difficulties.

Verapamil is known to interact with the MDR efflux pump, and Hendrikseet al. have demonstrated that images can be obtained with ¹¹C-verapamilin rats. Hendrikse N H, de Vries E G, Eriks-Fluks L, van der Graaf W T,Hospers G A, Willemsen A T, Vaalburg W, Franssen E J. A new in vivomethod to study P-glycoprotein transport in tumors and the blood-brainbarrier. Cancer Res. 1999; 59:2411–6. Their work also showed thatmodulation of MDR in vivo could be demonstrated in rats with¹¹C-verapamil. Although ¹¹C-verapamil may be an elegant probe for MDRper se, it is neither structurally nor functionally related to thetaxanes, anthracyclines, anthracenediones, camptothecin analogs, or anyother approved anticancer drugs. It is also important to recognize that,in humans, there is rapid and extensive catabolism of verapamil.Schomerus M, Spiegelhalder B, Stieren B, Eichelbaum M. Physiologicaldisposition of verapamil in man. Cardiovasc. Res. 1976; 10(5):605–12.Verapamil itself constitutes only a small fraction of the circulatingradioactivity, so the interpretation of the total radioactivity signalobtained with PET is problematic.

Also, although daunorubicin has been used as a probe in cell culture,where it is a stable molecule, it is not a stable molecule in the bodyand imaging with this component is not very useful. In humans,daunorubicin is converted rapidly by carbonyl reductase to its alcoholmetabolite, daunorubicinol. Thus, the signal observed via external PETimaging of labeled daunorubicin is a mixture of these two chemicalentities, which can complicate the interpretation. The plasma ratio ofmetabolite-to-parent is about 2.5:1 (Galettis P, Boutagy J, M a D D.Daunorubicin pharmacokinetics and the correlation with P-glycoproteinand response in patients with acute leukaemia Br. J. Cancer 1994;70(2):324–9), so it is possible that the metabolite is the speciesprimarily being imaged. However, it is also possible that tissue uptakeis more favorable for the parent, so a different ratio might be found.

⁹⁹Tc-Sestamibi, which is routinely used for cardiac imaging, has alsobeen explored for tumor imaging. As in the case for verapamil, neitherthe structural nor functional properties of sestamibi resemble those foranticancer drugs. The clinical imaging results are mixed. This probeseems to have the ability to detect tumors and monitor response in someclinical settings. Mankoff D A, Dunnwald L K, Gralow J R, et al.Monitoring the response of patients with locally advanced breastcarcinoma to neoadjuvant chemotherapy using ^(99m)Tc-sestamibiscintimammography. Cancer 1999; 85(11):2410–23. It is reported that theefflux rate of ^(99m)Tc-sestamibi correlates with antitumor response, atleast for one stage of breast cancer. Ciarmiello A, Del Vecchio S,Silvestro P, et al. Tumor clearance of technetium 99m-sestamibi as apredictor of response to neoadjuvant chemotherapy for locally advancedbreast cancer. J. Clin. Oncol. 1998; 16:1677–83. However, in lungcancer, no predictive value was found. Sasaki M, Kuabara Y, Yoshida T,et al. Can 99m-Tc-mibi-SPECT predict the treatment response of lungcancer? J. Nucl. Med. 2000; 41:286P.

The invention provides advantages that were not previously available byproviding a non-invasive method for determining potential drug efficacyby measuring actual drug accumulation in tumors. This invention allowsfor the efficient determination of the potentially most efficacioustreatment plan for antitumor therapy by allowing for individualizedoptimization of drugs and dosage.

The invention offers the additional, previously unrealized advantage ofdeveloping individualized antitumor therapies specific to a particularpatient with a particular type of tumor in a particular stage ofdevelopment.

The invention also provides a method for determining the effectivenessof particular drugs as treatment for particular cancers over a broadrange of the population.

SUMMARY OF THE INVENTION

Non-invasive, external imaging methods to visualize the location of adrug in the body avoid the need for biopsies and also have thecapability of scanning large areas of the body, indeed, the entire bodyif necessary.

Since a drug is only effective if it reaches the desired site of action,a method to determine the location of the drug after administrationallows monitoring of the potential effectiveness of drug administration.Antitumor drugs in particular are often toxic, and monitoring couldreduce the time required to find the most effective and targetedtherapy. Administration of a radio-labeled antitumor drug (preferably,the chemotherapeutic drug itself) which has been radio-labeled,preferably with a positron emitter, can provide external monitoring ofdrug accumulation in the tumor and/or normal host tissue by use ofimaging technologies, preferably with a PET scanner.

The present invention describes a method of labeling antitumor drugswith positron emitters, preferably the radioactive label ¹¹C. Theinvention further describes the use of labeled antitumor drugs in thetreatment of solid tumors. The invention is also a method of usinglabeled antitumor drugs to monitor the accumulation of drugs to solidtumors and to monitor modulators of cellular accumulation of drugs whichact on mechanisms in the cancer cells of the tumors that prevent uptakeand retention of drugs.

In one aspect, the invention is a method of measuring the accumulationof antitumor drugs by solid tumors comprising, administering anantitumor drug labeled with a positron-emitter to a patient having asolid tumor, and imaging at least part of the patient using PET.

Typically, solid tumors include breast, lung, ovarian, gastrointestinal,prostate, sarcoma and head and neck tumors. The measurement ofaccumulation of the drug may be measured either before or during aparticular treatment regimen, and may be used to determine the efficacyof an antitumor drug for treating solid tumors; to measure theeffectiveness of modulators of cellular accumulation includingmodulators of efflux and influx mechanisms; to measure the effectivenessof a combination of antitumor drugs where one or more of the drugs islabeled and the drugs are administered either simultaneously orsequentially; and for any other purpose for which measuring cellularaccumulation of an antitumor would be beneficial. Preferred drugs foruse in the invention include ¹¹C-paclitaxel, ¹¹C-docetaxel,¹¹C-doxorubicin, ¹¹C-epirubicin, ¹¹C-mitoxantrone, and ¹¹C-topotecan,although any drug for the treatment of solid tumors in radio-labeledform may be used.

In another aspect, the invention is a composition which is aradio-labeled drug. In particular, radio-labeled taxanes, especially¹¹C-paclitaxel and ¹¹C-docetaxel; radio-labeled anthracyclines,especially ¹¹C-doxorubicin and ¹¹C-epirubicin and other radio-labeledantitumor drugs, especially ¹¹C-topotecan. The invention is also amethod of preparing radio-labeled drug. In particular, radio-labeledtaxanes, especially ¹¹C-paclitaxel and ¹¹C-docetaxel; radio-labeledanthracyclines, especially ¹¹C-doxorubicin and ¹¹C-epirubicin and otherradio-labeled antitumor drugs, especially ¹¹C-topotecan.

The scope of this invention covers the preparation and diagnostic usesof antitumor drugs including paclitaxel, docetaxel, doxorubicin,epirubicin and others labeled with a positron emitter, preferably ¹¹C.Based upon the images obtained, the likelihood of success for treatmentof a patient's tumor with antitumor drugs can be predicted, as well asthe utility of any modulators of drug delivery.

One goal of the present invention is to avoid exposing patients to toxicdrugs that have no potential for benefit. The screening procedure of thepresent invention allows a rapid determination of whether a given tumorwill be likely to respond to a particular drug. Such a rapiddetermination is important for other reasons, including the tendency oftumors to become refractory with time. Thus, immediate imaging with¹¹C-paclitaxel, ¹¹C-docetaxel, ¹¹C-doxorubicin, ¹¹C-topotecan,¹¹C-mitoxantrone or other radio-labeled antitumor drugs has theadvantage of selecting patients with tumors that are likely to respondto therapy with the particular drug that is used. Further, the impact ofvarious doses and schedules for delivery can be monitored in situ at theactual tumor under treatment conditions. This is important since levelsof MDR and other modifiers of drug efficacy are often induced or alteredduring the course of therapy. It is within the scope of the presentinvention to re-test patients as often as therapeutically ordiagnostically necessary to assess the changing efficacy of treatment.

The method of the present invention involves administration of tracer,nontoxic quantities of an antitumor drug that has been radio-labeledwith a positron emitter. Imaging technologies such as a PET (PositronEmission Tomograph) scanner, can provide the ability to externallymonitor drug delivery to the tumor and/or normal host tissue. It isparticularly noteworthy that this procedure is noninvasive and can beused in a prognostic sense, i.e., before a particular drug isadministered in therapeutic quantities.

Preferred drugs for use in the invention are docetaxel, paclitaxel,doxorubicin, epirubicin, mitoxantrone and topotecan that have beenradio-labeled with a positron emitter. Although a wide range of labelingtechniques are known, for example replacing H with ¹⁸F, theradiolabelled drug is preferably identical to the non-labeled drugexcept that it contains a radio-labeled atom. Thus, preferredradio-labeled drugs are those in which a naturally occurring atom isreplaced with the same atom. Most preferably, ¹²C is replaced with ¹¹C.

Others have used radio-labeled drugs to explore drug distribution in thebody, but the present invention is specifically useful in measuring drugaccumulation in a tumor in order to make therapeutic decisions. Forexample, accumulation of a drug in a tumor may be measured to makepredictions regarding effectiveness of the drug in a particular patientor against a particular tumor. Similarly, accumulation of an antitumordrug may be measured when the drug is co-administered with anotherantitumor drug or with a cellular accumulation modifier. Significantly,the present invention measures drug accumulation regardless of themechanism of drug delivery and regardless of any particular transportpump (influx or efflux) that may be operating.

Further objectives and advantages will become apparent from aconsideration of the description and examples.

DETAILED DESCRIPTION OF THE INVENTION

In describing preferred embodiments of the present invention, specificterminology is employed for the sake of clarity. However, the inventionis not intended to be limited to the specific terminology so selected.All references cited herein are incorporated by reference as if each hadbeen individually incorporated.

The term “a” is intended to mean at least one unless the contextindicates otherwise.

Imaging Method According to the Invention

The present invention describes a method of visualizing antitumor drugsin the body. The drugs are first labeled by inserting a positronemitting isotope in the drug and administered the drug to a patient.Preferably, the drug is labeled by replacing a normally occurring atomwith a radioactive atom (e.g., ¹¹C for ¹²C or ¹⁸F for ¹⁹F) in the drugstructure. The antitumor drugs labeled with positron-emitting isotopesare then visualized by means of PET scan of the host's body. For thepurpose of the present invention, a host includes any animal, preferablya human, to whom the positron emitting drugs of the present inventionare administered for any therapeutic, diagnostic, prognostic, orexperimental purpose.

The object of labeling antitumor drugs is to determine and monitor thelocation of the drug in the body by imaging. For example, fortaxane-type drugs, the invention may utilize ¹¹C-paclitaxel or¹¹C-docetaxel. When cells accumulate antitumor drugs labeled with apositron-emitting isotope, the location of tissues comprising the cellscan be determined by a PET scan.

According to the invention, a positron-labeled anti tumor drug can beadministered simultaneously with the non-radio-labeled antitumor drug tocustomize treatment under the actual conditions of use. Positron-labeleddrugs may also be administered in test quantities to determine thelikelihood that the drug would be an effective therapeutic.

Significantly, many antitumor drug metabolites are rapidly excreted inthe bile, so that circulating radioactivity would be primarily theparent molecule.

Need for Exact Match of Drug and Imaging Profile

Within the limits of our present understanding of antitumor drugpharmacology, only the radio-labeled versions of the drugs themselvescan be relied upon to predict accurately therapeutic drug delivery. Theuse of any other molecule, no matter how apparently similar, potentiallycompromises the quality of the whole concept.

Recently, Li et al. have attempted to label paclitaxel with indium. LiC, Yu DF, Inoue T, Yang D J, et al. Synthesis, biodistribution andimaging properties of indium-111-DTPA-paclitaxel in mice bearing mammarytumors. J Nucl. Med. 1997;38:1042–7. These investigators-usedDiethylenetriaminepentaacetic acid anhydride (DTPA), a chleating agent,to attach ¹¹¹In to paclitaxel. The distribution in the body was found tobe significantly different for the ¹¹¹In-DTPA-paclitaxel than for theparent paclitaxel and was particularly affected by renal excretion.Because of these differences, it would require significantexperimentation to characterize and correlate the properties ofindium-DTPA-paclitaxel with paclitaxel.

In addition to the above concerns, both ¹¹¹In-DTPA and ^(99m)Tc areinferior atoms for imaging compared with positron-emitters such as ¹¹Cand ¹⁸F. Unlike ¹¹¹In-DTPA and ^(99m)Tc, positron emitters ultimatelygenerate two coincident photons, which permits the three-dimensionalposition within the body to be determined. The sensitivity of positronemitters such as ¹¹C is considerably greater, which permits smallertumors to be imaged.

In contrast to the substitution of non-radio-labeled atoms withradio-labeled atoms, attaching “extra” atoms to the drug, or changingsome of the atoms in the drug, creates an unacceptable situation, in theabsence of additional information. Using taxanes as an example,replacement of a hydrogen atom with longer-lived isotopes of fluorine oriodine has been attempted.

Ojima et al. report considerable differences in tumor cell sensitivitywhen fluorine analogs of docetaxel are evaluated, particularly at the3′-C locus. Ojima I, Inoue T, Chakravarty S. Enantiopurefluorine-containing taxoids: potent anticancer agents and versatileprobes for biomedical problems. J. Fluorine Chem. 1999; 97:3–10. Mostimportantly, the largest differences were observed for cell linesresistant to docetaxel. This variation in responsiveness might lead tosome promising leads for alternative drug discovery. However, it wouldbe misleading if the fluorinated molecule was used as a probe fordecision-making regarding treatment with the parent docetaxel, because afavorable outcome would be projected in situations in which the tumorwas actually resistant to docetaxel.

An iodinated version of paclitaxel has recently been proposed as aprobe. John C S, Cole C E, Kiesewetter D O, Eckelman W C. Synthesis,characterization, in-vitro cell binding, and biodistribution ofradioiodinated [125-I]-paclitaxel. J. Nucl. Med. 2000; 41:229P. Althoughlimited biodistribution data were reported, no attempt was made todemonstrate that it was comparable to the parent paclitaxel molecule. Infact, the iodine was placed on the phenyl ring of the benzoyl groupattached to the amino moiety at the 3′-C position of paclitaxel. This isthe same portion of the molecule which produced variations forfluorinated analogs of docetaxel. It's also the major site of structuraldifference for docetaxel versus paclitaxel: substituting a tert-butylgroup for the phenyl moiety.

Thus, considerable caution is required for any such modification. Theburden of evidence that such a change is acceptable must be very high,particularly since the imaging information will be used to guidedecisions about therapy of patients with life-threatening disease.

In addition, the short half-life of ¹¹C (20 minutes) is advantageouswhen compared to the 6 hour half-life for ^(99m)Tc. Thus, multipleinterventions may be conducted and probed in sequence during a singleclinic day with ¹¹C labeled drug, whereas ^(99m)Tc labeled drug would belimited to one procedure per day, due to carryover of radioactivity fromprior dosing. The shorter half-life of ¹¹C also substantially reducesthe biohazard, compared with ^(99m)Tc, since most of the injectedradio-activity would have left the patient before the patient left theclinic. Importantly, while expensive and extensive preclinicaltoxicology studies are usually required prior to human testing of a drugvariant, none are required for positron-labeled version at a tracer doseof a currently-marketed drug. Radiodosimetry studies, of course, arestill required.

In the case of taxanes, only paclitaxel and docetaxel are currentlyapproved for therapeutic use. Other taxanes are under development, andmight be approved and marketed in the future. If so, then these wouldalso be candidates for PET imaging. Thus, nothing in this disclosure isintended to limit the application of these concepts solely to taxanessuch as ¹¹C-docetaxel or ¹¹C-paclitaxel. Similarly, doxorubicin andepirubicin are the major anthracyclines for solid tumor therapy, butthese concepts are not limited to ¹¹C-doxorubicin or ¹¹C-epirubicin. Itis obvious to one skilled in the art that other classes of antitumordrugs may also be used in practicing the invention.

Assessment of Therapeutic Effects of Drugs on Particular Tumor Types

In an anticancer drug development program, the general strategy is tofind the tumor types most likely to respond to a particular antitumordrug. Direct demonstration of clinical benefit is critical. Usingtypical methods, these definitive studies require hundreds or thousandsof patients, which is resource-intensive and takes time. It takes timeto determine the success or failure in a general tumor type, and thus,to determine whether to continue investing money in clinical trials.

The invention may be used to assess the likelihood of success in using aparticular antitumor drug as a treatment for a particular type of tumor.According to the invention, a radio-labeled antitumor drug may beadministered to patients having a particular tumor type. Theaccumulation of the radio-labeled drug in the tumors can be measuredusing the method of the invention. Because analysis of drug accumulationin the tumor may be measured directly and results will not be dependenton measuring the long term response of patients, the number of patientsrequired to conduct studies predicting drug effectiveness and the amountof time required to analyze the results may both be markedly reduced. Inaddition, the effects of modulators of cellular accumulation of drugsand the effectiveness of co-administration of more than one antitumordrug (as described below) may be addressed in the context of the broaderpatient population.

Individualization of Therapy for Patients

Because there are multiple reasons for drug accumulation failures, asuccessful interventional strategy generally requires knowledge of themost important factor for each situation. However, administration of adrug labeled with a positron emitter allows for monitoring of thesuccess of modulation to be determined non-invasively with externalimaging techniques, if the radio-labeled agent is the same as the drugitself (e.g., paclitaxel), regardless of the mechanism(s) of drugdelivery failure. Accordingly, if a tumor is known or suspected to beresistant to docetaxel, paclitaxel, doxorubicin, epirubicin, or anotherantitumor drug, imaging with a positron-labeled version of the drug candetermine if the failure to respond is due to inadequate accumulation.

Recently, researchers have found that the MDR transporter is one of manytransporters and other similar mechanisms, which prevent theaccumulation of drugs in cells. For example, there are a large family ofATP-binding cassette (ABC) transporters, including MDR, MRP, and others.Expression of such transport mechanisms will vary from tumor-to-tumor.If accumulation of the drug is determined to be inadequate, possibly dueto the presence of such systems, one course of action that is underintensive research is the search for strategies to improve drugaccumulation in the tumor, potentially converting the situation from afailure to a treatment success.

Because it is known that the failure to accumulate drugs in the tumor inadequate amounts is a major cause for treatment failure, intensiveresearch efforts are underway to improve drug delivery and/or retention.Modulators of cellular accumulation mechanisms are often discovered inthe laboratory which increase the accumulation of antitumor drugs intumor cells. The present invention allows rapid determination of theusefulness of such a modulator for specific patients with positronlabeled antitumor drugs, by directly assessing whether drug accumulationis improved. Thus, the invention may be used to assess the impact ofvarious modulators, doses, and schedules for accumulation in situ at theactual tumor to be treated.

Antitumor drugs have also been given intraperitoneally for treatment ofovarian carcinoma, with encouraging results. The ability to measure drugconcentration at the tumor and/or normal tissue is critical to theevaluation of the success or failure of the therapy. Thus, the inventionprovides a means for directly assessing drug accumulation, a markedadvantage over simply measuring plasma concentrations. Using theinvention, it is thus possible to adjust therapy to fit a particularclinical situation. For example, the effect of drug uptake and retentionmodulators on drug delivery can be directly measured.

Assessment of Modulating Strategies

One of the major determinants of cellular accumulation is the operationof the efflux pumps. Many antitumor drugs may be effluxed from cellswherein MDR or other efflux pumps or transporters are highly expressed.The drugs are effluxed before they can accumulate. For example, taxanesmay be prevented by efflux pumps from reaching their target,microtubules, and therefore do not bind to the microtubules. Thus, thepresence of efflux transporters can prevent drugs from therapeuticaction in the cell. Efflux transporters will also prevent accumulationof the radio-labeled antitumor drugs. In cells with highly expressedefflux mechanisms, antitumor drugs do not accumulate. The failure toaccumulate antitumor drugs by tumor cells can indicate that high levelsof efflux pumps are expressed in these cells and preventing therapeuticlevels from being attained.

For commonly-used chemotherapy drugs such as taxanes, anthracyclines andvinca alkaloids, many tumor cells are resistant because they preventaccumulation of the drug into the cell. A common mechanism for thisfailure to accumulate a drug is the efflux pump MDR In order to addressthese difficulties, modulators of cellular accumulation mechanisms(modulators) may be administered with an antitumor drug to increase drugaccumulation in the tumor. By monitoring changes in accumulation when amodulator is added, the effectiveness of the modulator may bedetermined.

Other accumulation systems, for example, influx pumps or transporters,also exist and accumulation is the balance between influx and effluxrates. One skilled in the art will appreciate that similar principlesapply to both efflux and influx systems. Thus, the present invention maybe applied to influx pumps as well.

A variety of attempts to inhibit MDR have entered clinical testing,including dexverapamil, PSC833 from Novartis, LY335979 from Lilly, GG918from Glaxo and VX-853 from Vertex. Some surfactants, such as the solventCremophor®, which is used in the intravenous formulation of Taxol®, havebeen reported to be modulators of paclitaxel uptake and retention bytumor cells. The present invention may be utilized to monitor theeffectiveness of these and other cellular accumulation modulators, suchas the modulation of efflux mechanisms by any modulator including use ofexcipients used in formulations of drugs.

Current clinical investigations of MDR modulators have only measuredplasma concentrations of the drug itself. Since the purpose ofmodulation is to selectively improve accumulation of the drug to thetumor, the plasma data are not definitive. Because paclitaxel anddocetaxel are substrates for MDR, studies directed towards clarifyinguptake of these drugs by tumor cells would be clinically useful.Measurement of drug concentration in sequential biopsies of the tumor,with and without modulator, could provide more definitive comparisons,but are not practical in a clinical situation. However, according to theinvention, ¹¹C-paclitaxel or ¹¹C-docetaxel can be used to directlyassess the success or failure of these MDR-modulating strategies at thetumor itself. Similarly, doxorubicin and epirubicin are substrates formultiple efflux pumps, so that determination of the accumulation of¹¹C-doxorubicin or ¹¹C-epirubicin would be clinically useful. The methodaccording to the present invention utilizes positron-labeled antitumordrugs to measure the efficacy of MDR modulators of cancer cell efflux,influx, or other accumulation mechanisms.

The invention also envisions that substances which moderate or reduceefflux mechanisms could be co-administered with the radio-labeledantitumor drugs. Thus, it would be possible to determine an optimal doseof such a moderator that would allow therapeutic levels of antitumordrugs to accumulate in cells. Since the current list of known effluxpump moderators is quite low, and many are only known to work incell-culture and not necessarily in vivo, the invention encompasses theuse of labeled antitumor drugs as a research tool to find and exploreother efflux moderators.

In addition to MDR, modulating strategies can be directed at othertargets, including some which might be directed at decreasing normalhost tissue concentrations as a way of reducing patient toxicity, ratherthan increasing tumor concentration. Overall, the same goal is sought:to increase selective action of the drug at the tumor site underconditions tolerable to the patient.

The present invention is useful in determining the level of impact ofefflux type pumps or transporters on drug accumulation and therebywhether antitumor drugs are likely to be effective in the tumor imaged.When a radio-labeled antitumor drug is administered to a patient with atumor with low drug accumulation, the tumors cannot be imaged by a PETscan, indicating that the tumor is unlikely to be effectively treated bythe antitumor drug.

Assessment of Combination Chemotherapy Strategies

Often, when antitumor drugs are used therapeutically, maximal effectsare achieved by co-administering more than one drug, such as thecombination of a taxane with doxorubicin. The invention uses labeledantitumor drugs as a tool to determine optimal combinations of drugsthat are co-administered. The determination of which drugs toco-administer will depend on the clinical circumstances of the diseaseto be treated. The decision of whether to co-administer a drug and whichdrug to use will depend on the clinical decision of the practitioner.

Although the combination of taxanes with doxorubicin is very promising,only plasma pharmacokinetics are currently accessible to assesstherapeutic strategy Gianni, L. et al. Human pharmacokineticcharacterization and in vitro study of the interaction betweendoxorubicin and paclitaxel in patients with breast cancer. J. Clin.Oncol. 15:1906–12, 1997; Gianni, L et al. Paclitaxel by 3-hour infusionin combination with bolus doxorubicin in women with untreated metastaticbreast cancer: high antitumor efficacy and cardiac effects in a dose-and sequence-finding study. J. Clin. Oncol. 13:2688–2699, 1995.) Ananalogous set of clinical trials with docetaxel and variousanthracyclines are underway. Sparano J A, O'Neill A, Schaefer P L, etal. Phase II trial of doxorubicin and docetaxel plus granulocytecolony-stimulating factor in metastatic breast cancer: EasternCooperative Oncology Group Study E1196. J. Clin. Oncol. 2000;18:2369–77.Assessing the accumulation of ¹¹C-taxane can play a major role in doseand sequence decisions for these trials.

The present invention contemplates co-administration of radio-labeledantitumor drugs to determine efficacy of such combinations. Thus, usingthe imaging method of the invention, the effect of co-administration ofmore than one antitumor drug on drug accumulation may be determined.This determination may be made by assessing the accumulation of one orboth of the antitumor drugs which are co-administered. Accordingly, oneor both of the co-administered antitumor drugs is labeled with a radioisotope and the accumulation of the radio-labeled drug at the tumor ismeasured using PET techniques.

Synthesis of Positron Emitting Antitumor Drugs

In another aspect, the invention also relates to certain radio-labeledantitumor drugs useful in practicing the invention and methods ofsynthesizing radio-labeled drugs. In particular, the invention is amethod for synthesizing radio-labeled taxanes and anthracyclines. Theinvention is not limited to the specific drugs and methods of theexamples, but include any equivalents to those compositions andprocedures as would be suggested to one of skill in the art.

Taxanes

In particular, the invention includes compounds having the formula:

where R₁ is selected from the group consisting of H, acetate and¹¹C-acetate; R₂ is selected from the group of acetate and ¹¹C-acetate;R₃ is selected from the group consisting of benzoyl, ¹¹C-benzoyl and—CO₂C(CH₃)₃ and —¹¹CO₂C(CH₃)₃; R₄ is selected from the group consistingof benzoyl and ¹¹C-benzoyl, and wherein the compound contains at leastone atom of ¹¹C. Radio-labeled paclitaxel and docetaxel are preferredtaxanes. Radio-labeled paclitaxel may have a ¹¹C labeled acetate in the4-position or the 10-position, or may have a radio-labeled benzoyl groupat the R₃ amide or at R₄ in the 2-position. Radio-labeled docetaxel mayhave a radio-labeled tert-butyl carboxyl group at R₃, acetate at R₂ orbenzoyl at R₄. Typically, the acyl groups are labeled at the carbonylcarbon.

The procedures for the synthesis of radio-labeled taxanes aremodifications of those reported by Rao, K V et al. (Synthesis andevaluation of some 10-mono- and 2′,10-diesters of 10-deacetylpaclitaxel.J Med. Chem. 38:3411–14, 1990), and by Murray et al. in U.S. Pat. No.5,808,113. The present methods utilize different reagents and reactionconditions as compared to these reports. The differences improve theefficiency of the reaction with respect to addition of ¹¹C, i.e., viaacetylation or benzoylation.

From the perspective of a large-scale manufacturing process,10-deacetylpaclitaxel or the other taxane precursors are scarce andexpensive resources, and the procedures are optimized for high yields ofthe taxane starting material. In this regard they are successful; e.g.,Rao et al. report 85% conversion of 10-deactylpaclitaxel to paclitaxel.However, Rao et al. used approximately a 200-fold a molar excess ofacetyl donor. Based upon acetyl groups, the yield of this reaction wouldbe 0.5%, which is impractical for radio-synthesis of an ¹¹C-labeled PETprobe. The present invention accomplishes the higher yields of 10–20%(based on acetyl donor) required to make PET imaging feasible. Thus, thepresent invention represents a major improvement over Rao et al. Usingroutine experimentation, a person skilled in the art may make minoralterations in the synthesis as may be required to adapt the conditionsto those routinely used at their particular radiotracer facility.

Verification of the identify of the product is obtained by comparisonwith authentic nonradioactive reference material of paclitaxel(available commercially, e.g., from Sigma Chemical Company, HauserChemical Company, HandeTech Corporation) or docetaxel (available fromAventis/RPR). The final product, e.g., ¹¹C-paclitaxel or ¹¹C-docetaxel,is purified (e.g., using a solid-phase extraction cartridge) andprepared for intravenous injection in a suitable solvent (e.g., normalsaline, Cremophor®, ethanolic solution, Tween 80).

Similar methods may be useful for preparing other radio-labeled taxanesby incorporation ¹¹C into ester or carbamate substituents on thebaccatin nucleus or side chains.

Anthracyclines

The invention also includes radio-labeled anthracyclines andparticularly radio-labeled doxorubicin and epirubicin having the generalformula:

wherein one of A is H and B is OH (¹¹C-doxorubicin) or A is OH and B isH (¹¹C-epirubicin).

Due to the lack of commercial availability of a suitable anthracyclineantitumor drug precursor for radiolabeling, several initial steps arerequired to generate a precursor for preparing radio-labeled doxorubicinand related drugs. In essence, after protecting the amine group in thesugar and the —OH groups, the methyl group at the 4-O-position isremoved, producing a suitable precursor. Because these steps areperformed prior to radiolabeling, high yield and rapid reactions are notessential. Radiolabeling of the precursor can be accomplished with¹¹C-methyl iodide, a standard radiolabeling reagent. Deprotectionproduces the desired radio-labeled product.

Other Antitumor Drugs

The invention also includes other radio-labeled anti-tumor drugs andparticularly radio-labeled topotecan having the general formula:

Radiolabeled topotecn may be prepared from N-desmethyl topotecan, whichhas been reported in the literature (Rosing H, Herben V M M, vanGortel-van Zomeren D M, et al, Isolation and structural confirmation ofN-desmethyl topotecan, a metabolite of topotecan, Cancer Chemotherapyand Pharmacology (1997) 39:498–504), but is not commercially available.The N-desmethyl topotecan is prepared from commercial topotecan througha procedure adapted from the procedure of Rao, et.al; (Rao, P. N.,Acosta, C. K., Cessac, J. W., Bahr, M. L., Kim, H. K. Synthesis ofN-desmethyl derivatives of 17a-acetoxy-11b-(4-N,N-dimethylaminophenyl)-19-norpregna4,9-diene-3,20-dioneand mifepristone. Steroids 1999; 64:205–212. The ¹¹C-topotecan isprepared from N-desmethyl topotecan by methylation with ¹¹C-methyliodide.

Another potentially useful radio-labeled drug for use in practicing theinvention is ¹¹C-mitoxantrone. Current efforts for preparing this drugby placing a ¹¹C-label in one of the hydroxyethyl groups have not beensuccessful.

EXAMPLES Example 1 Synthesis of ¹¹C-Paclitaxel

10-Deacetylpaclitaxel (6 mg) (commercially available) in pyridine (0.5ml) was reacted with chlorotriethylsilane (0.1 ml) for 1 hour at 60° C.to yield 7,2′-di-(triethylsilyl)-10-deacetylpaclitaxel. This molecule isthe immediate precursor for radiolabeling; it is stable at roomtemperature and can be stored until needed.

To a solution of (50 μg, 60 nmol) of7,2′-di-(triethylsilyl)-10-deacetylpaclitaxel, was addeddimethylaminopyridine solution (90 μl of 300 mg/ml in methylenechloride), and of tert-butyl diphenyl chlorosilane (20 μL). Acetylchloride (10 μl of a 1:5000 solution in methylene chloride, 25 nmol) wasthen added and the mixture heated to 105° C. for 10 minutes. The silylgroups were removed within 2 minutes by adding of methylene chloride(400 μl) and tetrabutylammonium fluoride solution (50 μl of 1 M solutionin tetrahydrofuran). Verification of the identify of the product viaHPLC and mass spectrometry was obtained by comparison with authenticreference material of paclitaxel (available commercially). The overallyield, based upon the acetyl donor, was 10–20%.

This procedure works with sufficient yield (10–20% overall), in therequired time limit (15 minutes from addition of acetyl chloride untilbeginning of clean-up), and with small quantities of material (e.g., 50μg). Thus, ¹¹C-paclitaxel may be prepared simply by substituting¹¹C-acetyl chloride for unlabeled acetyl chloride.

On the day of use, ¹¹C is prepared by a cyclotron in the form of¹¹C—CO₂. This radio-labeled carbon dioxide is rapidly converted to¹¹C-acetyl chloride using published procedures. Luthra S K, Pke V W,Brady F. Preparation of some NCA [1-11-C] acid chlorides as labellingagents. Appl. Radiat. Isot. 1990; 41:471–6. The final product,¹¹C-paclitaxel, is purified (e.g., using a solid-phase extractioncartridge or HPLC) and prepared for intravenous injection in a suitablesolvent (e.g., normal saline, Cremophor® or ethanolic solutions).

Example 2 Alternative Synthesis of ¹¹C-Paclitaxel

Benzoyl chloride (8 μl of 1:1000 dilution in acetonitrile) is added to asolution of paclitaxel primary amine (50 μg) (commercially-available) inacetonitrile (200 μl). After 2 minutes at room temperature, 60% recoveryof product was obtained.

Conversion of this procedure to radio-synthesis requires preparation of¹¹C-benzoyl chloride, which is known in the art by following typicalGringard reaction schemes. Mathews W B, Burns H D, Danals R F, Rabert HT, Naylor E M. Carbon-11 labeling of a potent, nonpeptide, AT1-selectiveangiotensin-II receptor antagonist, MK-996. J. Labelled Compounds andRadiopharmaceuticals 1995; XXXVI:729–37. For ¹¹C-paclitaxel,purification may be accomplished by HPLC or by adding acidic water andmethylene chloride.

Example 3 Synthesis of ¹¹C-Docetaxel

A. Preparation of Docetaxel Primary Amine

The starting material for the preparation of ¹¹C-docetaxel is docetaxelprimary amine, which is not available commercially. The free amine isprepared by removal of the tert-butyl carbonyl group from docetaxelusing formic acid or other methods known in the art.

Docetaxel was purified from its commercial form (TAXOTERE® for InjectionRhonePoulencRorer) by silica gel chromatography. Docetaxel (4.5 mg) wasdissolved in EtOH (1 ml). After addition of formic acid (1 ml) themixture was stirred at room temperature for 10 days. Overall conversionto docetaxel primary amine was 48%. Water (2 ml) and methylene chloride(2 ml) were added to the reaction solution and mixed. The aqueous phasecontaining docetaxel primary amine was separated from the organic phasecontaining residual docetaxel. The aqueous phase was neutralized withsaturated sodium bicarbonate in water. Methylene chloride (12 ml) wasadded to the aqueous component, mixed and centrifuged. The organicphase, which contained the docetaxel primary amine, was separated anddried with a gentle stream of air at 55° C.

B. Preparation of ¹¹C-Docetaxel

Docetaxel primary amine (100 μg) was dissolved in ethyl acetate (200 μl)and 0.6 equivalents of di-tert butyl dicarbonate (di-BOC) was added. Thesolution was heated at about 60° C. for 10 minutes. The docetaxelproduct was produced in 15% yield and its identity verified by HPLC.

This rapid, single-step process for preparation of unlabeled docetaxelmay be converted to a radioactive method by the use of ¹¹C-labelleddi-BOC. The key step is substitution of ¹¹C—CO₂ for unlabeled CO₂.Preparation of di-tert-butyl dicarbonate is described in severalpublications, including: U.S. Pat. No. 5,151,542, entitled, “Process ForPreparing Di-Tert-Butyl Dicarbonate” or U.S. Pat. No. 5,162,565,entitled, “Process For Preparing Ditertiary-Alkyl Dicarbonate”.

Example 4 Alternative Synthesis of ¹¹C-Docetaxel

In a two-step procedure, 10-acetyldocetaxel is prepared from thepaclitaxel primary amine, followed by 10-deacetylation via hydrogenperoxide to yield docetaxel. Paclitaxel primary amine (0.5 mg) was addedto ethyl acetate (0.2 ml) containing an equimolar amount of di-tertbutyl dicarbonate. The solution was heated at 65° C. for 30 minutes toform 10-acetyl docetaxel. Upon heating at 125° for about 6 minutes, 20%conversion of 10-acetyl-docetaxel to docetaxel was obtained in 15%hydrogen peroxide.

As in the preceding example, conversion of this procedure toradio-synthesis requires preparation of ¹¹C-labeled di-BOC.

Example 5 Synthesis of ¹¹C-Doxorubicin

A. Preparation of Protected Demethylated Doxorubicin

Protected demethylated doxorubicin, the precursor for the synthesis ofradio-labeled doxorubicin was formed by first dissolving doxorubicin (20mg, 36 μmol) in isopropanol (5 ml) and sodium borate (1 ml of 0.25M),and reaction with 9-Fluorenylmethyl chloroformate (fmoc) (20 mg, 70μmol) at room temperature with sonication for 20 min. The solution wasreduced in volume under a stream of air to ˜1 ml, extracted intochloroform, and washed with water (2×). The chloroform was removed undera stream of air, leaving dox-fmoc suitable for the next step.

Dox-fmoc (10 mg) was dissolved in dry pyridine (3 ml) and cooled in anice bath. Benzoyl chloride (125 μl, 0.9 mmol) was slowly added, andallowed to react for 30 min. The reaction (monitored by HPLC) wasquenched by addition of water (0.5 ml), and blown down under air (50°C.). The reaction mixture was extracted into methylene chloride andwashed with water (3×). The methylene chloride was removed under astream of air, and dried under vacuum, producing dox-fmoc-4bz asidentified by HPLC-MS.

Dox-fmoc-4bz was dissolved in methylene chloride (about 4 ml) and cooledin a dry ice/isopropanol bath. BCl₃ (100–200 μl of 2M in heptane) wasadded. The reaction was maintained at −78° C. for about 2 hours(monitored by LC), and quenched by addition of 2% acetic acid (1 ml) at−78° C., washed with water (2×), and the methylene chloride removedunder a stream of air, and dried under vacuum. The 4-OH-dox-fmoc-4bzproduct was isolated by HPLC, in about 5% yield.

B. Synthesis of ¹¹C-Doxorubicin

Methylation conditions were adapted from Bernardi et al. Bernardi L,Masi P, Spaini O, Suarato A, Arcamone F.4-Demthoxy-4-alkoxydaunorubicins. II Farmaco Ed.Sc. 1978; 34:884–889.The 4-OH precursor (4-OH-dox-fmoc-4bz) was added to a vial containingAgO (approximately 3 mg) and isopropanol (250 μl). Methyl iodide (10 ug,75 nmol) was introduced, the vial was capped and heated at 120° C. forabout 13 minutes. The reaction mixture was filtered and dried under astream of air. The protecting groups were removed stepwise, adapting theprocedures of Adams. Adams N, Blake C, Broadhurst M J et al. Synthesisand antitumor activity of novel 4-demethoxyanthracyclines. J Med. Chem.1990; 33:2375–9. The dry reaction products were dissolved in acetone towhich 0.5 volumes of A dilute KOH is added, heated to 100° C. for about6 minutes, then neutralized with dilute acetic acid and dried. Thereaction products are then redissolved in chloroform and treated withmorpholine at 100° C. for 5 minutes. Doxorubicin can then be isolated byHPLC.

Radio-labeled doxorubicin is prepared by substituting ¹¹C-methyl iodidefor the nonradioactive methyl iodide in the methylation step. Oneskilled in the art will appreciate that the reaction has been conductedon a scale and with amounts of reagents in the range and within atimeframe suitable for tracer radio labeling with ¹¹C-methyl iodide.Preparation of ¹¹C-methyl iodide is established in the art and astandard reagent at facilities equipped for PET synthesis.

¹¹C-epirubicin may be prepared in an identical manner by substitution ofepirubicin for doxorubicin.

Example 6 Synthesis of ¹¹C-topotecan

A. Synthesis of the N-Desmethyl Topotecan.

The starting material for the preparation of ¹¹C-topotecan, N-desmethylTopotecan, is not commercially available. The N-desmethyl topotecan isprepared from commercial topotecan through a procedure adapted from theprocedure of Rao, et. al. (Rao, P. N., Acosta C. K., Cessac, J. W.,Bahr, M. L., Kim, H. K. Synthesis of N-desmethyl derivatives of 17a-acetoxy-11b-4-N,N-dimethylaminophenyl)-19-norpregna4,9-diene-3,20-dioneand mifepristone. Steroids 1999; 64:205–212.

To a solution of Topotecan dissolved in 2:1 tetrahydrofuan:methanol(v:v) was added approximately 20 mol eq. CaO, and cooled in an ice bath.10 mol eq. of I₂ was added. The reaction was allowed to proceed for 2hours with periodic vortexing. The reaction was quenched with 10% sodiumthiosulfate, acidified with dilute formic acid, the solids removed bycentrifugation, and dried. N-Desmethyl topotecan was purified by HPLC.Identity of the N-desmethyl topotecan was confirmed by LC/MS.

B. Preparation of ¹¹C-Topotecan

N-desmethyl topotecan was dissolved in dimethylforamide to which methyliodide was added. The reaction vessel was capped and heated at 100° C.,for about 4 minutes. The reaction mixture was concentrated and¹¹C-Topotecan purified by HPLC.

Radio-labeled topotecan is prepared by substituting ¹¹C-methyl iodidefor nonradioactive methyl iodide. Preparation of ¹¹C-methyl iodide isestablished in the art and a standard reagent at facilities equipped forPET syntheses.

Example 7 General Procedures for Clinical Use

The positron-labeled antitumor drug is prepared shortly before use,i.e., within 2 hours of injection, and preferably within less than 1hour. The positron-labeled antitumor drug with an activity of 1 to 300mCi, preferably 10–60 mCi, is injected into the patient as anintravenous bolus, i.e., within less than 5 minutes, preferably in 1minute. The patient is placed in a PET scanner, and images are obtainedusing standard techniques known to the art at 5- to 10-minute intervalsfollowing the injection, up to at least 60 minutes, preferably 90minutes, when image quality is satisfactory. When ¹⁸F or otherpositron-emitting isotopes with a longer half-life is used, imaging forhours or a day or more may be feasible. Variations and improvements inmachine technology may permit even longer imaging periods, which isdesirable. It is within the scope of the present invention for anoperator skilled in the art of PET scanning to modify the method in suchways as changes and improvements in PET scans require or allow.

Example 8 Use of Positron-Labeled Antitumor Drugs for Selection ofChemotherapy

A patient with a tumor is imaged with one or more positron-labeledantitumor drugs according to the procedures in Example 7. One or more ofthe labeled drugs is injected. If more than one antitumor drug is used,a 90 minute period (or longer) between injections is necessary for ¹¹Clabeled drugs. Four or more hours between separate injections may berequired if 18F or other isotopes with longer half-lives are used. Basedupon the accumulation of antitumor drug demonstrated in the images,therapy can be guided by whether the patient's tumor is classified assensitive or resistant to one or more of the drugs.

Example 9 Development of Modulators for Antitumor Drug Delivery toTumors

The procedures in Example 7 can be used to obtain a baseline evaluationof antitumor drug accumulation within the tumor, in the absence of anymodulation attempts. These procedures are then repeated in the presenceof a specific modulation strategy, or a series of modulation attempts,and the images are compared to determine success or failure.

Example 10 Combinations of Antitumor Drugs

The procedures used in Example 7 may be used to monitor antitumor drugaccumulation in both normal tissue and tumors when combinations ofantitumor drugs are used under a variety of dose and temporaladjustments. In practicing the method, one of the antitumor drugs islabeled in order to determine the effect of other antitumor drugs on thedelivery of the labeled drug. Alternatively, if the drugs areadministered sequentially and non-simultaneously, both of the antitumordrugs of the combination may be labeled in order to determine drugdelivery overall.

Example 11 Targeting of Tumor Types for Drug Development

The use of positron-labeled antitumor drugs, following the procedures ofExample 7, provide a basis for selecting tumor types for furtheremphasis in drug development. Indeed, the entire paradigm of drugselection could shift from dominance by histopathologic findings toreliance upon actual accumulation of radio-labeled antitumor drugs toguide initial therapy.

The embodiments illustrated and discussed in this specification areintended only to teach those skilled in the art the best way known tothe inventors to make and use the invention. Nothing in thisspecification should be considered as limiting the scope of the presentinvention. All examples presented are representative and non-limiting.The above-described embodiments of the invention may be modified orvaried, and elements added or omitted, without departing from theinvention, as appreciated by those skilled in the art in light of theabove teachings. It is therefore to be understood that, within the scopeof the claims and their equivalents, the invention may be practicedotherwise than as specifically described.

1. A method of measuring the accumulation of anti-tumor drugs by solidtumors comprising, administering an anti-tumor drug labeled with apositron-emitter to a patient having a solid tumor, and imaging at leastpart of the patient using PET, wherein said anti-tumor drug is aninsoluble taxane.
 2. The method according to claim 1, wherein the solidtumor is selected from the group consisting of breast, lung, ovarian,gastrointestinal, prostate, sarcoma and head and neck tumors.
 3. Themethod of claim 1, wherein the labeled drug is at least one drugselected from the group consisting of ¹¹C-paclitaxel and ¹¹C-docetaxel.4. A method of determining the efficacy of an anti-tumor drug fortreating solid tumors comprising: administering an anti-tumor druglabeled with a positron-emitter to a patient having a solid tumor; andimaging at least part of the patient by PET to measure accumulation ofthe labeled anti-tumor drug, wherein said anti-tumor drug is aninsoluble taxane.
 5. The method according to claim 4, wherein thelabeled anti-tumor drug is administered prior to a course of treatmentof the patient.
 6. The method of claim 4, wherein the labeled anti-tumordrug is administered during the course of treatment of the patient. 7.The method of claim 4, wherein the labeled drug is at least one drugselected from the group consisting of ¹¹C-paclitaxel and ¹¹C-docetaxel.8. A method of measuring the effectiveness of modulators of cellularaccumulation mechanisms in tumors comprising: administering ananti-tumor drug labeled with a positron-emitter to a patient;administering a modulator to the patient, and imaging at least part ofthe patient by PET to measure accumulation of the labeled anti-tumordrug, wherein said anti-tumor drug is an insoluble taxane; theaccumulation of labeled anti-tumor drug is measured before and afteradministering the modulator to the patient; and the levels of anti-tumordrug accumulation before and after administering the modulator arecompared.
 9. The method of claim 8, wherein modulator affects an effluxmechanism.
 10. The method of claim 8, wherein modulator affects aninflux mechanism.
 11. The method of claim 8, wherein the labeled drug isat least one drug selected from the group consisting of ¹¹C-paclitaxeland ¹¹C-docetaxel.
 12. A method for measuring the effectiveness of acombination of anti-tumor drugs comprising: administering more than oneanti-tumor drug to a patient having a solid tumor, wherein at least oneof said anti-tumor drugs is labeled with a positron-emitter, and imagingat least part of the patient by PET to measure accumulation of the atleast one said anti-tumor drug labeled with a positron-emitter, whereinthe at least one said anti-tumor drug labeled with a positron-emitter isan insoluble taxane.
 13. The method of claim 12, wherein two anti-tumordrugs are administered to the patient.
 14. The method of claim 12,wherein said labeled anti-tumor drugs is labeled with apositron-emitter.
 15. The method of claim 12, wherein two of saidanti-tumor drugs are each labeled with a positron-emitter.
 16. Themethod claim 12, wherein a first anti-tumor drug and a second anti-tumordrug are administered simultaneously.
 17. The method claim 12, wherein afirst anti-tumor drug and a second anti-tumor drug are administeredsequentially.
 18. The method of claim 12, wherein the labeled drug is atleast one drug selected from the group consisting of ¹¹C-paclitaxel and¹¹C-docetaxel.
 19. A compound having the formula:

wherein: R₁ is selected from the group consisting of H, acetate and¹¹C-acetate; R₂ is selected from the group of acetate and ¹¹C-acetate;R₃ is selected from the group consisting of benzoyl, ¹¹C-benzoyl,—CO₂C(CH₃)₃ and —¹¹CO₂C(CH₃)₃; and R₄ selected from the group consistingof benzoyl, ¹¹C-benzoyl; and wherein the compound contains at least oneatom of ¹¹C.
 20. A compound according to claim 19, wherein R₁ is¹¹C-acetate, R₂ is acetate, R₃ is benzoyl and R₄ is benzoyl.
 21. Acompound according to claim 19, wherein R₁ is acetate, R₂ is ¹¹C-acetateand R₃ is benzoyl and R₄ is benzoyl.
 22. A compound according to claim19, wherein R₁ and R₂ are acetate and R₃ is ¹¹C-benzoyl and R₄ isbenzoyl.
 23. A compound according to claim 19, wherein R₁ and R₂ areacetate, R₃ is benzoyl and R₄ is ¹¹C-benzoyl.
 24. A compound accordingto claim 19, wherein R₁ is H, R₂ is acetate, R₃ is —¹¹CO₂C(CH₃)₃, and R₄is benzoyl.
 25. A compound according to claim 19, wherein R₁ is H, R₂ is¹¹C-acetate, R₃ is CO₂C(CH₃)₃ and R₄ is benzoyl.
 26. A compoundaccording to claim 19, wherein R₁ is H, R₂ is acetate, R₃ is —CO₂C(CH₃)₃and R₄ is ¹¹C-benzoyl.
 27. A method of synthesizing the compoundaccording to claim 19, comprising the steps of: reacting10-deacetylpaclitaxel with a chlorotrialkylsilane to yield a protecteddeacetylpaclitaxel; reacting the protected deacetylpaclitaxel with¹¹C-acetyl chloride to yield a radio-labeled silyl protecteddeacetylpaclitaxel; removing the protecting groups, and isolating¹¹C-paclitaxel.
 28. A method of synthesizing the compound according toclaim 19, comprising the steps of: reacting paclitaxel primary aminewith ¹¹C-benzoyl chloride, and isolating ¹¹C-paclitaxel.
 29. A method ofsynthesizing the compound according to claim 19, comprising the stepsof: reacting docetexal primary amine with ¹¹C-di-tert-butyl dicarbonate,and isolating ¹¹C-docetaxel.
 30. A method of synthesizing the compoundaccording to claim 19, comprising the steps of: reacting paclitaxelprimary amine with ¹¹C-di-tert-butyl dicarbonate to give¹¹C-10-acetyldocetaxel; and reacting the ¹¹C-10-acetyldocetaxel withhydrogen peroxide to give ¹¹C-docetaxel.
 31. A method of measuring theaccumulation of anti-tumor drugs by solid tumors comprising,administering an anti-tumor drug labeled with a positron-emitter to apatient having a solid tumor, and imaging at least part of the patientusing PET; wherein said anti-tumor drug labeled with a positron-emittercomprises an insoluble taxane having a naturally occurring atom replacedwith a radioisotope of the same element.
 32. A method of measuring theaccumulation of anti-tumor drugs by solid tumors comprising,administering an anti-tumor drug labeled with a positron-emitter to apatient having a solid tumor, and imaging at least part of the patientusing PET; wherein the anti-tumor drug comprises a compound having theformula:

wherein: R₁ is selected from the group consisting of H and acetate; R₂is acetate; R₃ is selected from the group consisting of benzoyl and—CO₂C(CH₃)₃; and R₄ is benzoyl, wherein the compound contains at leastone atom of ¹¹C.
 33. The method of claim 32, wherein R₁ is ¹¹C-acetate,R₂ is acetate, R₃ is benzoyl and R₄ is benzoyl.
 34. The method of claim32, wherein R₁ is acetate, R₂ is ¹¹C-acetate and R₃ is benzoyl and R₄ isbenzoyl.
 35. The method of claim 32, wherein R₁ and R₂ are acetate andR₃ is ¹¹C-benzoyl and R₄ is benzoyl.
 36. The method of claim 32, whereinR₁ and R₂ are acetate, R₃ is benzoyl and R₄ is ¹¹C-benzoyl.
 37. Themethod of claim 32, wherein R₁ is H, R₂ is acetate, R₃ is —¹¹CO₂C(CH₃)₃,and R₄ is benzoyl.
 38. The method of claim 32, wherein R₁ is H, R₂ is¹¹C-acetate, R₃ is CO₂C(CH₃)₃ and R₄ is benzoyl.
 39. The method of claim32, wherein R₁ is H, R₂ is acetate, R₃ is —CO₂C(CH₃)₃ and R₄ is¹¹C-benzoyl.
 40. A method of measuring the effectiveness of modulatorsof cellular accumulation mechanisms in tumors comprising: administeringan anti-tumor drug labeled with a positron-emitter to a patient;administering a modulator to the patient, and imaging at least part ofthe patient by PET to measure accumulation of the labeled anti-tumordrug, wherein said anti-tumor drug is an insoluble taxane and themodulator affects tumor concentration of the anti-tumor drug or normalhost cell concentration of the anti-tumor drug; the accumulation oflabeled anti-tumor drug is measured before and after administering themodulator to the patient; and the levels of anti-tumor drug accumulationbefore and after administering the modulator are compared.
 41. Themethod of claim 40, wherein the modulator affects the activity of atleast one of an efflux pump or transporter and an influx pump ortransporter.
 42. The method of claim 40, wherein the modulator changesthe baseline normal host cell accumulation of the anti-tumor drug. 43.The method of claim 40, wherein modulator is an MDR modulator.
 44. Themethod of claim 41, wherein modulator is selected from dexverapamil,PSC833, LY335979, GG918, VX-853, Cremophor® and surfactants.
 45. Themethod of claim 40, wherein the labeled drug is at least one drugselected from the group consisting of ¹¹C-paclitaxel and ¹¹C-docetaxel.